In vitro activity of tedizolid against 43 species of Nocardia species

The purpose of the present study was to evaluate the in vitro activity of tedizolid against several clinically significant species of Nocardia by comparing with that of linezolid. A total of 286 isolates of Nocardia species, including 236 clinical isolates recovered from patients in Japan and 50 strains (43 species) purchased from NITE Biological Resource Center, were studied. Antimicrobial susceptibility testing was performed using the broth microdilution method. For the 286 Nocardia isolates, the minimal inhibitory concentration (MIC)50 and MIC90 values of tedizolid were 0.25 and 0.5 μg/ml, and those of linezolid were 2 and 2 μg/ml, respectively. The distribution of the linezolid/tedizolid ratios (MICs of linezolid/MICs of tedizolid) showed that tedizolid had four- to eight-fold higher activity than linezolid in 96.1% (275/286) of Nocardia isolates. Both the tedizolid and linezolid MIC90 values for Nocardia brasiliensis were two-fold higher than those for the other Nocardia species. Both tedizolid and linezolid had low MIC values, 0.25–1 μg/ml and 0.5–4 μg/ml, respectively, even against nine isolates (five species) that were resistant to trimethoprim/sulfamethoxazole. One Nocardia sputorum isolate showed reduced susceptibility to tedizolid (4 μg/ml). Bioinformatics analysis suggests different resistance mechanisms than the oxazolidinone resistance seen in enterococci and staphylococci.


Bacterial isolates and identification
A total of 286 strains of Nocardia species, including 236 clinical isolates (15 species/complexes) recovered from patients in 27 microbiology laboratories in Japan and 50 strains (43 species) purchased from the NITE Biological Resource Center (NBRC) (National Institute of Technology and Evaluation, Tokyo, Japan), were studied (Table 1).Identification of Nocardia species was based on Gram-stain, colonial morphology, and molecular technique.Species identification of all clinical isolates was performed by Matrix-Assisted Laser Desorption/Ionization Timeof-Flight Mass Spectrometry (microflex LT, Bruker Daltonics) 15 and by full-length 16S rRNA gene sequencing 16 .Nocardia spp. that were difficult to identify via 16S rRNA gene sequencing were classified as a complex 17,18 .Two strains of new species of N. sputorum (IFM 12275, IFM 12276 T ), which were previously reported as a novel species by our group 19 , were included in the N. abscessus complex.These strains were closely related to N. beijingensis NBRC16342 T (99.6%) by phylogenetic analysis based on the 16S rRNA sequence 19 .The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and the complete genome sequence of strains IFM 12276 T and IFM 12275 are LC741024 and AP026978, and LC741023 and AP026976 (plasmid: AP026977), respectively.

Antimicrobial susceptibility testing (AST)
Antimicrobial Susceptibility Testing (AST) of 286 Nocardia isolates was performed using the broth microdilution method with frozen panels (Eiken Chemical, Tokyo, Japan), according to the Clinical and Laboratory Standards Institute (CLSI) M24-A3 guidelines 20 .In brief, a heavy organism suspension was prepared in a small volume of sterile saline with 7-10 3-mm glass beads and was vortexed vigorously.Clumps were allowed to settle for 15 min, and the supernatant was adjusted to a 0.5 McFarland standard using a calibrated nephelometer.For frozen panel inoculation, the adjusted 0.5 McFarland suspension was diluted 30-fold with sterile saline, and 10 µl of the diluted solution was dispensed into each well of the panel.The panels were incubated at 35 °C for 48-72 h and, if needed, daily thereafter for up to 5 days.For trimethoprim/sulfamethoxazole, the MICs were determined as the wells corresponding to 80% inhibition of growth compared to the controls.The MICs were determined for tedizolid, linezolid, trimethoprim/sulfamethoxazole, amikacin, tobramycin, clarithromycin, amoxicillin-clavulanic acid, ciprofloxacin, moxifloxacin, minocycline, imipenem, and ceftriaxone on the same panel, and were interpreted as recommended by CLSI.For this analysis, Staphylococcus aureus ATCC 29213 and Nocardia nova ATCC BAA-2227 were used as the quality control strains.
For determination of trimethoprim/sulfamethoxazole resistance, disk diffusion testing with a 250-μg sulfisoxazole disk (Hardy Diagnostics, CA, USA) was performed according to the CLSI M24-A3 guidelines 20 .For this analysis, N. nova ATCC BAA-2227 and Escherichia coli ATCC 25922 were used as the quality control strains.

Minimum bactericidal concentration testing
Minimum bactericidal concentration (MBC) testing for tedizolid and linezolid against 23 clinical isolates (eight of Nocardia farcinica complex, four of Nocardia cyriacigeorgica, three of N. brasiliensis, three of N. nova complex, two of Nocardia abscessus complex, two of Nocardia transvalensis complex, and one of Nocardis sp.) was performed according to the Clinical Microbiology Procedures Handbook 21 .The isolates were selected from species that are frequently isolated from clinical materials.The MBC was defined as the lowest antibiotic concentration able to kill at least 99.9% (reduction of 3 log10) of the initial inoculum.

Ethical statement
The present study was conducted in accordance with the ethical guidelines of the Ministry of Health, Labour and Welfare, Japan.No ethical committee approvals or informed consent were needed for this study.
Table 1.Species distribution of the 286 Nocardia strains subjected in this study.a NITE Biological Resource Center (NBRC), b N. farcinica and N. kroppenstedtii were included in the N. farcinica complex, c N. nova, N. elegans, N. aobensis, and N. veterana were included in the N. nova complex, d N. abscessus, N. asiatica, N. beijingensis, N. sputorum, and N. arthritidis were included in the N. abscessus complex, e N. wallacei, N. transvalensis, and N. blacklockiae were included in the N. transvalensis complex.N. nova complex c 37

Antimicrobial Susceptibility Testing
The MIC ranges, MIC 50 values, MIC 90 values and MIC distributions for tedizolid and linezolid against the 286 isolates of Nocardia species/complexes are shown in Table 2 and Fig. 1.For the 286 Nocardia isolates, the MIC 50 and MIC 90 values of tedizolid were 0.25 and 0.5 μg/ml, and those of linezolid were 2 and 2 μg/ml, respectively.Both the tedizolid and linezolid MIC 90 values for N. brasiliensis were two-fold higher than those for the other Nocardia species.Both tedizolid and linezolid had low MIC ranges, 0.25-1 (mode = 0.5) μg/ml and 0.5-4 (mode = 2) μg/ml, respectively, even against the nine isolates that were resistant to trimethoprim/sulfamethoxazole isolates (three of N. otitidiscaviarum, two of N. transvalensis complex, two of N. mexicana, one of N. cyriacigeorgica, and one of N. terpenica).On the other hand, one clinical isolate of N. abscessus complex (N.sputorum IFM 12275) indicated higher MIC of tedizolid (4 μg/ml, measured three times on different days) than the other isolates (up to 1 μg/ml).
The distribution of the linezolid/tedizolid ratios (MICs of linezolid/MICs of tedizolid) against the 286 isolates of Nocardia species are shown in Fig. 2. Tedizolid was four-to eight-fold more active than linezolid in 96.1% (275/286) of Nocardia isolates.One clinical isolate of N. abscessus complex (N.sputorum IFM 12275) showed the same MIC value for linezolid (4 μg/ml) as that for tedizolid.

Antimicrobial susceptibility patterns for the different Nocardia species
Antimicrobial susceptibility patterns for the different Nocardia species consisting of 236 clinical isolates and 50 NBRC strains are shown in Table 3.Only three drugs, amikacin, linezolid, and trimethoprim/sulfamethoxazole, showed ≥ 90% susceptibility among the all 286 isolates.The antimicrobial susceptibility patterns varied by species, Table 2. MIC ranges, MIC 50 and MIC 90 values for tedizolid and linezolid against the 286 isolates of Nocardia species.a N. farcinica and N. kroppenstedtii were included in the N. farcinica complex, b N. africana, N. aobensis, N. elegans, N. kruczakiae, N. nova, N. vermiculata and N. veterana were included in the N. nova complex, c N. abscessus, N. arthritidis, N. asiatica, N. sputorum, and N. beijingensis were included in the N. abscessus complex, d N. blacklockiae, N. transvalensis, and N. wallacei were included in the N. transvalensis complex.www.nature.com/scientificreports/and multidrug resistance (defined as four or fewer drugs that exhibit a susceptibility rate of 90% or higher) was common among N. farcinica complex, N. nova complex, N. abscessus complex, N. transvalensis complex, N. otitidiscaviarum, N. thailandica, N. mexicana, and N. concava.www.nature.com/scientificreports/

Minimum bactericidal concentration testing
The distribution of the MBC/MIC ratios of tedizolid and linezolid against the 23 clinical isolates of the seven Nocardia species showed that the MBC/MIC ratio was greater than or equal to eight for all 23 isolates.

Analysis of resistance mechanisms in tedizolid resistant isolate
The ARGannot analysis on IFM 12275 resulted in hits for ole(C), blab-4, aph(3″), tlr(C), and TlrC, while CARD analysis resulted in hits for vanY, vanW, and rifampin monooxigenase, but acquired linezolid resistance genes known in enterococci, cfr, cfr(B), cfr(D), optrA, and poxtA were not detected by either method.On the other hand, the genes that were hits in the IFM 12275 strain other than TlrC were also detected in the IFM 12276 T strain.Of note, the TlrC gene has been reported as a gene associated with resistance for macrolide, lincosamide, and streptogramin group antibiotics 30 .In addition, in silico analysis by the Primer-blast confirmed that the acquired linezolid resistance gene described above was not detected in IFM 12275.
In the analysis of nucleotide mutations in the 23S rRNA gene (G2576T, G2505A, U2500A, G2447U, G2534U, G2603U), no mutations were observed in either of IFM 12275 and IFM 12276 T strains.Furthermore, the 50S ribosomal protein sequences of L3 (rplC), L4 (rplD), and L22 (rplV) were the same between the two strains.Comparing the gene sets in IFM12275 with those in IFM12276 T , a total of 6285 orthologous genes were detected, and 500 and 387 unique genes were harboured in IFM12275 and IFM12276 T , respectively.

Discussion
Sulfonamides, mainly trimethoprim/sulfamethoxazole, have been the antimicrobials of choice to treat nocardiosis for a long time 4 .Trimethoprim/sulfamethoxazole still has a good activity against Nocardia species isolated in Japan 15 .However, adverse reactions to high-dose trimethoprim/sulfamethoxazole therapy, such as myelosuppression and hepatoxicity, are frequent 4 , and change in antibiotics is often necessary in such cases.Furthermore, the emergence of resistant bacteria due to prophylactic administration of low-dose trimethoprim/sulfamethoxazole has also been regarded as a problem.Averbuch et al. reported that the proportion of trimethoprim/sulfamethoxazole resistant Nocardia spp. was four of 25 (16%) among patients who received trimethoprim/sulfamethoxazole prophylaxis at the time of nocardiosis, compared with two of 36 (6%) among those who did not 2 .
Tedizolid has bacteriostatic activity against gram-positive cocci by binding to the 23S ribosomal RNA of the 50S subunit of the bacterial ribosome and inhibiting the early steps of bacterial protein synthesis 31 .Compared to linezolid, tedizolid shows stronger binding at the site of activity due to its unique D-ring substituent 31 ; therefore, tedizolid is four-to 16-fold more potent in vitro than linezolid against many clinically relevant gram-positive cocci, including linezolid-resistant strains 26,32 .In the present study, we found that both tedizolid and linezolid had bacteriostatic activity (MBC/MIC ratio ≥ 8 21 ) against Nocardia spp., and tedizolid was four-to eight-fold Table 3. Antimicrobial susceptibility patterns for the different Nocardia species consisting of 236 clinical isolates and 50 NBRC strains.a N. farcinica and N. kroppenstedtii were included in the N. farcinica complex, b N. africana, N. aobensis, N. elegans, N. kruczakiae, N. nova, N. vermiculata and N. veterana were included in the N. nova complex, c N. abscessus, N. arthritidis, N. asiatica, N. sputorum, and N. beijingensis were included in the N. abscessus complex, d N. blacklockiae, N. transvalensis, and N. wallacei were included in the N. transvalensis complex, e Resistance to trimethoprim-sulfamethoxazole were determined by disk diffusion testing with a 250-μg sulfisoxazole disks.f S, susceptible (only one isolate), Bold in the table indicates a susceptibility rate of 90% or higher.www.nature.com/scientificreports/more active than linezolid in 96.1% (275/286) of Nocardia isolates.Brown-Elliott et al. reported that tedizolid had higher antibacterial activity in vitro than linezolid against 101 isolates of Nocardia species.They also cited the advantages of tedizolid over linezolid, including fewer serious adverse events, higher oral bioavailability, higher intracellular concentration, and a longer half-life, and speculated on its potential for the treatment of infections caused by Nocardia spp. 33.There are some clinical case reports of the successful use of tedizolid for the treatment of disseminated nocardiosis [34][35][36][37] .In the four cases outlined in those case reports, all patients were initially treated with antimicrobial agents including linezolid, but tedizolid was used as an alternative due to myelosuppression caused by linezolid.In all cases, the patients were safely treated with tedizolid on prolonged treatment for two to six months without the development of myelotoxicity [34][35][36][37] .However, some studies have suggested the risk of thrombocytopenia with tedizolid 38 ; further assessment is needed of thrombocytopenia development on prolonged treatment in the clinical setting.

Species (no. of isolates tested) % of susceptible isolates of species or antibiotic susceptible pattern Amikacin
On the other hand, in the present study, we found that one isolate, N. sputorum (IFM12275) showed higher MIC of tedizolid (4 μg/ml) than the other isolates (up to 1 μg/ml).The MIC of tedizolid for N. sputorum (IFM12275) was 16-fold higher than that for the same species, IFM12276 T .Interestingly, this isolate had the same MIC value for linezolid (4 μg/ml).In the CLSI method, there is an interpretive breakpoint (BP) of linezolid against Nocardia species at 8 μg/ml, but no BP of tedizolid.Considering the BPs of linezolid and tedizolid are 4 μg/ml and 0.5 μg/ml for S. aureus and 2 μg/ml and 0.5 μg/ml for Enterococcus faecalis, respectively, the Nocardia isolate with MIC of 4 μg/ml in the present study is thought to be resistant to tedizolid.The mechanism of resistance to linezolid and tedizolid is being studied mainly in staphylococci and enterococci [12][13][14]22 . In hese species, linezolid resistance is mediated through acquisition of resistance genes (cfr, etc.) or through ribosomal mutations in 23S rRNA gene or 50S ribosomal proteins L3, L4, and L22 12,22 .On the other hand, it has been reported that although tedizolid is active against linezolid-resistant stapylococci possessed cfr gene, it has cross-resistance to linezolid when mutations in chromosomal genes encoding 23S rRNA or ribosomal proteins (L3 and L4) are present 13,39 .Interestingly, it is also reported that the MICs of linezolid were four-to 16-fold higher than those of tedizolid, even in such cross-resistant strains to linezolid and tedizolid 12,14 , suggesting that the tedizolid-resistant strain in the present study with the same MIC values as linezolid, IFM 12275, is a very atypical strain.
Valdezate et al. reported two linezolid-resistant N. farcinica strains isolated from patients with cystic fibrosis 40 .These isolates indicated very high MIC of linezolid with ≥ 256 μg/ml.In their study, Valdezate et al. investigated the presence of genetic resistance determinants by PCR and a complete genome analysis of the strains, but they were unable to identify the resistance mechanism.Neither of the isolates harboured the cfr gene, nor did they show the mutations in 23S RNA (G2576T) allowing linezolid resistance, although the G2608A change was found in one allele of the one strain 41 .In the present study, we found that the tedizolid-resistant strain did not harbour the acquired linezolid resistance genes, nor did it show the mutations in 23S RNA and the 50S ribosomal protein.
These results indicate that oxazolidinone resistance in Nocardia spp. is caused by a different resistance mechanism reported in enterococci and staphylococci.Since 500 genes were uniquely harboured in the tedizolid-resistant strain compared with the tedizolid-susceptible strain, another tedizolid-resistant strain would be helpful in identifying the candidate genes.Unfortunately, in the present study, we were unable to elucidate the mechanism of resistance to tedizolid in Nocardia species.Further research is necessary to elucidate the resistance mechanism and to set interpretive BP for tedizolid against Nocardia species.
A multicentric retrospective cohort study by Takamatsu et al. reported that the most frequently isolated Nocardia species in Japan were N. farcinica (79/317, 24.9%), N. nova complex (61/317, 19.2%), N. abscessus complex (59/317, 18.6%), and N. cyriacigeorgica (44/317, 13.9%) 41 .Identification of clinical isolates of Nocardia to the species/complex level is important, because Nocardia spp.differ in clinical spectrum and susceptibility patterns 42 .In the present study, differences in drug susceptibilities and multidrug resistance patterns were also observed depending on the Nocardia species.Recently, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based identification has been identified as a rapid, easy, and reliable method 15,43,44 .Accurate identification by MALDI-TOF MS and antimicrobial susceptibility profiles together can help earlier implementation of empirical treatment and improvement of patient prognosis.On the other hand, we found one isolate which showed reduced susceptibility to tedizolid; in addition, linezolid-resistant clinical isolates have already been reported 40,45 , so performing antimicrobial susceptibility testing against all clinically significant isolates is recommended, especially in disseminated Nocardia infection.